Pro-angiogenic peptides and uses thereof

ABSTRACT

This present invention is directed to peptides, compositions, and methods for modulating endogenous cytokine expression in a subject. More specifically, the invention provides peptides useful in regulating the release of a specific pattern of cytokines that promote angiogenesis and/or can be used to modulate the immune system of a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

More than one reissue application has been filed for the reissue of U.S.Pat. No. 8,460,697 B2. The reissue applications are application Ser. No.14/734,785 (issued as U.S. Pat. No. RE46,425) and Ser. No. 15/278,016(the present application), all of which are reissue divisionalapplications of U.S. Pat. No. 8,460,697 B2. The present application is areissue divisional of application Ser. No. 14/734,785 (issued as U.S.Pat. No. RE46,425), which is an application for reissue of U.S. Pat. No.8,460,697 B2, which is related to and claims the benefit of U.S.Provisional Application No. 61/221,021 filed Jun. 26, 2009, and; U.S.Pat. No. 8,460,697 B2 is also a continuation-in-part of U.S. patentapplication Ser. No. 11/955,143 filed Dec. 12, 2007, which in turnclaims the benefit of U.S. Provisional Application No. 60/869,862 filedDec. 13, 2006, the contents of each of which are incorporated herein byreference thereto.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 1650 2,148 byte ASCII (text) file named“Seq_List” created on Jun. 25, 2010 Jun. 9, 2015.

FIELD OF THE INVENTION

The present invention relates to the fields of therapeutic peptides andendogenous cytokine modulation. More specifically the invention relatesto the fields of angiogenesis, wound healing, and/or immune systemmodulation.

BACKGROUND

Impaired circulation is an underlying aspect of manyclinically-manifested disorders including peripheral arterial disease(PAD), ischemic heart disease and chronic wounds. More than 24 millionpatients are afflicted with these conditions in the US, with 10 millionaffected by PAD alone. PAD is often a result of diabetes, whichcurrently affects one in three adults over the age of 40 and whoseincidence is expected to increase as a result of increasing obesity inthe general population. Chronic wounds affect more than 6.5 millionpatients each year and cause significant impairment of quality of life.Even with current treatments, about 35,000 limb amputations areperformed each year due to life-threatening ischemia.

Wound healing after injury occurs in three major stages. First is thehemostatic and inflammatory stage, which minimizes blood loss andrecruits specific cells to the site of injury. Platelets engage theinjured tissue, initiate clot formation and release growth factorsduring this first stage. In the second stage, recruited phagocytic cellssuch as macrophages and monocytes digest the injured tissue andangiogenic growth factors released by activated platelets, macrophages,and other cells bind to receptors on the surface of endothelial cells inpre-existing blood vessels. The endothelial cells then proliferate,migrate into a wound bed, and differentiate into arterial and venousvascular tissue. Finally, in a third remodeling stage, new blood vesselsmature by recruiting smooth muscle cells to stabilize the vasculaturearchitecture, whereupon blood begins to flow through the new bloodvessels.

Angiogenesis, the process of growth of new blood vessels, is anessential process in wound healing and for restoring blood flow totissues after injury. The discovery of growth factors that stimulatethis process has had a major influence on medical treatment of cripplingand life-threatening conditions resulting from loss of bloodcirculation. At least 20 growth factors have been identified thatstimulate angiogenesis. The growth factor most widely studied, and usedclinically, is the pro-angiogenic platelet-derived growth factor-BB(PDGF-BB). PDGF is released from many cell types including activatedplatelets, activated macrophages, endothelial cells, fibroblasts andtumor cells, and PDGF was approved by the FDA in December 1997 forclinical use as a topical agent for diabetic foot ulcers. A secondgrowth factor developed for clinical use is the vascular endothelialgrowth factor (VEGF). Growth factors of this type and biologicallyactive analogs are typically mid-sized proteins which can be produced byrecombinant techniques (e.g. in yeast), and activation of angiogenesisby growth factors is accomplished, at least in part, by stimulation ofcytokine production.

IL-8 is a cytokine that activates neutrophils and has potent chemotacticactivity on neutrophils and lymphocytes. The inflammatory event at thesite of infection or injury activates monocytes and macrophages, whichrelease IL-8. Inflamed endothelial tissue also releases IL-8, whichattracts neutrophils from blood into the tissue during the initial phaseof the defense mechanism. The consequence is a vicious cycle ofrecruitment of neutrophils in response to IL-8, damage to tissues, andmore production of IL-8 leading to deleterious inflammation as a sideeffect. In addition, neutrophils adhere to inflamed endothelial tissuesthrough integrins secreted from the cells, and ICAM-1 can stimulaterelease of the integrins to which neutrophils bind, thereby increasingthe level of deleterious inflammation at the site of injury evenfurther. Increased levels of certain types of clinically deleteriouscytokines, such as IL-8 and ICAM-1, at a site of infection or injury cantherefore cause deleterious side effects which can hinder the process ofhealing.

The healing of wounds in mammalian tissue may be enhanced by theapplication, either alone or in combination with a cytokine and/orgrowth factor, of certain neuropeptides such as Tachykinins, SubstanceP, Substance K, and the like as well as calcitonin gene-relatedpeptides. The use of such peptides for clinical applications has,however, been hampered by several problematic issues includingdeleterious side effects. Substance P, for example, is a known mediatorof pain impulses and its effects on wound healing have been known forseveral years. However, Substance P has also been shown to stimulateneurons to release factors that recruit inflammatory cytokines andneutrophils to the site of a wound, thereby causing pain andinflammation.

Therefore, the use of peptides or growth factors and their analogs astherapeutic agents for wound healing can be problematic for a number ofreasons, including efficacy, cost, and deleterious side effects such asinflammation. Information relevant to attempts to address one or more ofthese problems can be found in the following references: U.S. Pat. No.7,105,481, U.S. Patent Application No. 2007/0021342, and U.S. PatentApplication No. 2007/0154448. However, each one of these referencessuffers from one or more of the following disadvantages:

1. the requirement for expression of a polynucleotide containing thenucleotide sequence encoding the protein, which can complicateproduction and significantly increase costs;

2. the requirement for purification of expressed proteins from the otherproteins of the host cell, which can complicate production andsignificantly increase costs;

3. administration of angiogenic growth factors without the furtherability to activate phagocytes and thereby enhance efficacy orameliorate infection or other concomitant disorders;

4. administration of angiogenic growth factors without the furtherability to reduce inflammation, thereby reducing deleterious sideeffects; and

5. administration of a peptide which can stimulate the release offactors that recruit inflammatory cytokines and neutrophils to the siteof a wound, causing pain and inflammation.

Therefore, in light of the available treatments for promoting woundhealing by stimulating angiogenesis, there is a need to providepractical, cost-effective therapies that enhance or optimize chronicwound healing without causing deleterious side effects.

SUMMARY OF THE INVENTION

The present invention provides novel therapeutic peptides, and methodsfor their use to modulate endogenous cytokine expression in a subject,promote angiogenesis and/or modulate a subject's immune system. Thetherapeutic peptide consists of only 5 to 6 amino acids, and is selectedfrom the group consisting of:

X1-Q-X2-X3-X4-X5; and

X1-N-S-X3-X4-X5

-   wherein X1 is selected from the group consisting of N, V, W, and Y;-   X2 is selected from the group consisting of H, A, and P;-   X3 is selected from the group consisting of T, Q, and S;-   X4 is selected from the group consisting of P, Q, H, L, and Y; and-   X5 is selected from the group consisting of R and S, or is absent.

Preferred peptides include for example, WNSTL (SEQ ID NO:1), NQHTPR (SEQID NO:2), WNSTY (SEQ ID NO:5), YNSTL (SEQ ID NO:6), YQPSL (SEQ ID NO:7),VQATQS (SEQ ID NO:8), and VNSQH VSNQH (SEQ ID NO:9). Preferably, thepeptide is in substantially pure form. Typically it is desired that thepeptide be at least 80% pure by weight. In one embodiment the N-terminusmay also be acetylated for stability. It is also preferable that thepeptide is pro-angiogenic. In a preferred embodiment, the peptides ofthe invention comprise a peptide construct with at least two or morearms. The construct typically has a central framework and each armconsists of a core sequence linked to the central framework via alinker. Each core sequence of the peptide construct can be the same ordifferent. In a preferred embodiment, the core sequence is the same foreach arm of peptide construct. Furthermore, the core sequence ispreferably selected from the group of therapeutic peptides describedabove.

The present invention also provides therapeutic compositions comprisingat least one peptide of the invention and a pharmaceutically acceptablecarrier.

In yet another aspect of the invention, the invention provides a methodof modulating the cytokine expression in a subject. The methodpreferably comprises administering to a subject one or more peptides ofthe invention in an amount sufficient to increase the expression of atleast one beneficial endogenous cytokine and/or reducing expression ofat least one harmful cytokine.

The peptides of the invention can be specifically used to treat certaindiseases, and especially induce angiogenesis and wound healing in asubject. In certain embodiments, the peptide is administered in amountsufficient to stimulate angiogenesis in a subject with a wound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a molecular structure of one embodiment of thepresent invention, a multivalent peptide construct containing fourpeptides according to the invention, each of which is linked to acentral core structure via a linker sequence;

FIG. 2A illustrates the chemical structure of a peptide constructaccording to one embodiment of the present invention, the constructcontains four copies of the core sequence WNSTL (SEQ ID NO:1) linked toa branched central framework structure;

FIG. 2B illustrates the chemical structure of a peptide constructaccording to one embodiment of the present invention, the constructcontains four copies of the core sequence NQHTPR (SEQ ID NO:2) linked toa branched central framework;

FIG. 3 illustrates the structure of a reporter tag that can be added toa peptide according to the present invention, a C-terminal extensioncontaining a fluorescent dansyl group; and

FIG. 4 is a bar graph of data illustrating the stimulation of growth ofa tumor as the result of pro-angiogenic activity by one embodiment ofthe present invention, the quadravalent peptide construct containing thecore sequence WNSTL (SEQ ID NO:1) as illustrated in FIG. 2A.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

To provide a therapeutic agent with broad properties that stimulateangiogenesis, a process essential to wound healing and restoration ofcirculation to damaged tissues, the agent should enhance healing withoutinducing clinically deleterious side effects such as inflammation, andpreferably would act in concert with phagocytic activity to eliminatetissue debris and attenuate bacterial infections. The peptides of thepresent invention meet these goals by concomitantly inducing release ofbeneficial cytokines, inhibiting the release of deleterious cytokinesand stimulating the activity of phagocytic cells. Treatment with thepeptides of the present invention induces the healing process byproviding a sustained endogenous elevation of an array of beneficialcytokines, in contrast to the administration of a single exogenouscytokine.

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the invention. It will beunderstood, however, by those skilled in the art, that the structures,compositions, and methods are sometimes shown or discussed generally inorder to avoid obscuring the invention. In many cases, a description ofthe material and operation is sufficient to enable one to implement thevarious forms of the invention. It should be noted that there are manydifferent and alternative technologies and treatments to which thedisclosed inventions may be applied, and the full scope of theinventions is not limited to the examples that are described below.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The present invention comprises therapeutic angiogenic peptides,compositions of those peptides for administration to a subject in need,and methods to stimulate both angiogenesis and the immune system of asubject through the administration of compositions containing thosepeptides.

The single letter designation for amino acids is used predominatelyherein. As is well known by one of skill in the art, the single letterdesignations are as follows: A is alanine; C is cysteine; D is asparticacid; E is glutamic acid; F is phenylalanine; G is glycine; H ishistidine; I is isoleucine; K is lysine; L is leucine; M is methionine;N is asparagine; P is proline; Q is glutamine; R is arginine; S isserine; T is threonine; V is valine; W is tryptophan; Y is tyrosine.

The Inventors have identified a series of polypeptides that modulate therelease of specific cytokines and/or stimulate the immune response.These polypeptides may be particularly beneficial to stimulate woundhealing in an injured subject. In general, the advantage of thisinvention is the modulated release of specific cytokines and thestimulation of angiogenesis. Nonlimiting examples of cytokines includeimmunoregulatory proteins such as interleukins and interferons, whichare secreted by cells of the immune system and can affect the immuneresponse. More particularly, the present invention relates to a familyof synthetic peptides capable of inducing the release of beneficialcytokines which can therapeutically stimulate wound healing withoutinducing deleterious side effects.

Thus, in a first aspect, the present invention provides a therapeuticpeptide consisting of 5 to 6 amino acids in length. The therapeuticpeptide modulates cytokine expression and preferably is angiogenic.Advantageously, the therapeutic peptide may be reacted with aceticanhydride as well as other compounds to acetylate the N-terminus of thetherapeutic peptide. This acetylation stabilizes the peptide andtherefore is preferred.

The therapeutic peptides are preferably in a substantially purifiedform. As used herein, the term “substantially purified” refers tomaterial which is substantially or essentially free from componentswhich normally accompany it as found in its native state. When thematerial is synthesized, the material is substantially or essentiallyfree of cellular material, gel materials, culture medium, chemicalprecursors, contaminating polypeptides, nucleic acids, and otherchemicals. Generally, the isolated or synthesized peptide will comprisemore than 70% or 80% (dry weight) of all macromolecular species presentin the preparation. Preferably, the protein is purified to representgreater than 90% (dry weight) of all macromolecular species present.More preferably the protein is purified to greater than 95% (dryweight), and most preferably the protein is purified to essentialhomogeneity, wherein other macromolecular species are not detected byconventional techniques.

Preferred therapeutic peptides for this first aspect of the inventionare selected from the group consisting of:

X1×2×3×4×5×6

-   wherein X1 is selected from the group consisting of N, V, W, and Y,    or is absent;-   X2 is selected from the group consisting of Q, V, S, W, N, and Y;-   X3 is selected from the group consisting of H, A, S, N, Q, and P;-   X4 is selected from the group consisting of T, N, Q, S, and P;-   X5 is selected from the group consisting of P, Q, H, T, L, Y, S; and-   X6 is selected from the group consisting of R, S, H, L, Y, or is    absent.

More preferably,

-   X1 is selected from the group consisting of N, V, W, and Y;-   X2 is selected from the group consisting of Q, S, and N;-   X3 is selected from the group consisting of H, A, N, S, and P;-   X4 is selected from the group consisting of T, Q, and S;-   X5 is selected from the group consisting of P, Q, H, L, and Y; and-   X6 is selected from the group consisting of R and S, or is absent.

And still more preferably,

-   X1 is selected from the group consisting of V, W, and Y;-   X2 is selected from the group consisting of Q, S, and N;-   X3 is selected from the group consisting of N, S, and P;-   X4 is selected from the group consisting of T, Q, and S;-   X5 is selected from the group consisting of H, L, and Y; and-   X6 is absent.

In an alternative preferred embodiment, the therapeutic peptide isselected from the group consisting of:

X1-Q-X2-X3-X4-X5; and

X1-N-S-X3-X4-X5

-   wherein X1 is selected from the group consisting of N, V, W, and Y;-   X2 is selected from the group consisting of H, A, and P;-   X3 is selected from the group consisting of T, Q, and S;-   X4 is selected from the group consisting of P, Q, H, L, and Y; and-   X5 is selected from the group consisting of R and S, or is absent.

More preferably,

-   X1 is selected from the group consisting of V, W, and Y;-   X2 is selected from the group consisting of A and P;-   X3 is selected from the group consisting of T and S;-   X4 is selected from the group consisting of Q and Y;-   X5 is S or absent.

Still more preferably,

-   X1 is selected from the group consisting of W and V;-   X2 is A;-   X3 is T;-   X4 is selected from the group consisting of L and Y;-   X5 is S or absent.

In a most preferred embodiment, the therapeutic peptide is selected fromthe group consisting of WNSTL (SEQ ID NO:1), NQHTPR (SEQ ID NO:2), WNSTY(SEQ ID NO: 5), YNSTL (SEQ ID NO:6), YQPSL (SEQ ID NO:7), VQATQS (SEQ IDNO:8), and VNSQH VSNQH (SEQ ID NO:9).

In a second aspect, the present invention provides a therapeuticangiogenic peptide comprising a construct and at least two arms. Theconstruct has a central framework and each arm consists of a coresequence linked to the central framework via a linker. Each coresequence can be the same or different.

As used herein, “construct” is defined as the entire molecule andcomprises the central framework linked with at least two arms. In apreferred embodiment, the construct comprises the central frameworklinked to 2 or more arms, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms,preferably 2 to 8 arms. In a further preferred embodiment, the constructcomprises the central framework linked to 4 arms. Each arm within theconstruct may consist of the same or different core sequence and/orlinker. In one preferred embodiment, the core sequence is the samebetween arms.

The “central framework” is defined as the structural core of theconstruct, providing a structure for attaching the arms to a centralstructure. The central framework is based on a core molecule which hasat least two functional groups to which molecular branches havingterminal functional groups are bonded, e.g., a tri-lysine to which thepeptide arms. Such molecules may be developed or created to present avarying number of branches, depending on the number of monomers branchedfrom the core molecule. Each non-terminal functional group on eachbranch provides a means of attachment to an arm. Non-limiting examplesof preferred central framework include: ethylenediamine(1,2-ethanediamine), ethylene glycol (1,2-dihydroxyethane), polyols suchas glycerol, 3,5-diaminobenzoic acid, 1,3,5-triaminobenzene, andmonocarboxylic-diamino compounds of intermediate length. Preferably, themonocarboxylic-diamino compounds are within the range of 2 to 10 carbonsin length. Non-limiting examples of such compounds are2,3-diaminopropionic acid and 2,6-diaminocaproic acid. In a morepreferred embodiment, the monocarboxylic-diamino compound is 6 carbonsin length. Compounds that provide an aromatic central framework whichabsorbs light may be beneficial for determining peptide concentration aswell. The carboxyl group of the monocarboxylic-diamino compounds allowsthe addition of C-terminal tags including biotin derivatives. In apreferred embodiment, the central framework comprises a tri-lysine core(a lysine residue as the core molecule bonded to two lysine residues,each through its carboxyl group, to one of the amino groups of thecentral lysine residue), providing a central frame work for up to fourarms.

The “arm” is defined as the core sequence, defined below, plus thelinker. The “linker” is defined as a peptide chain or other moleculethat connects the central framework to the core sequence. In a preferredembodiment, the linker comprises, but is not limited to, certain linkerpeptide sequences, polyethylene glycol, 6-aminocaproic acid(6-aminohexanoic acid), 8-aminooctanoic acid, and dextran. In a mostpreferred embodiment, the linker is GGGS (SEQ ID NO:3), GGGSGGGS (SEQ IDNO:4), SSSS (SEQ ID NO:10), SSSSSSSS (SEQ ID NO:11), or variationsthereof. The length of the linker can be adjusted, for example, thelinker GGGS (SEQ ID NO:3) can be repeated to provide variable lengths,e.g., repeated twice (GGGSGGGS (SEQ ID NO:4)), or even three or moretimes; additional serine residues could be added to SSSS (SEQ ID NO:10)to also produce varying lengths of the linker.

The “core sequence” is defined as the functional portion of each armthat provides the therapeutic effect. The core sequence is preferablyselected from the group of therapeutic peptides of 5 to 6 amino acids inlength described above in the first aspect. In a most preferredembodiment, the core sequence is selected from the group consistingspecifically of: WNSTL (SEQ ID NO:1), NQHTPR (SEQ ID NO:2), WNSTY (SEQID NO:5), YNSTL (SEQ ID NO:6), YQPSL (SEQ ID NO:7), VQATQS (SEQ IDNO:8), and VNSQH VSNQH (SEQ ID NO:9).

A specific illustration of a therapeutic peptide of the invention is setforth in FIG. 1. In FIG. 1, the therapeutic peptide is in the form of amultivalent immuno-regulatory peptide construct 10. The construct 10 canbe synthesized with at least two arms 1, (e.g., two, three, four, eightor more arms 1). The same core peptide sequence 2 can be used for eacharm or, alternatively, two or more different core peptide sequences 2can be used for each arm 1 instead. The length of the linker 3 betweenthe central framework 4 of the construct 10 and the core peptidesequence 2 determines the length of each of the arms 1. The arms 1illustrated in FIG. 1, for example, are often about 3±0.5 nm in lengthdepending on conformation, or approximately 7±0.5 nm across themolecule. Cell-surface domains of known receptor proteins arecorrespondingly about 3 to 4 nm in diameter. This distance can beadjusted by increasing or decreasing the length of the linker 3.Preferably, the length of each of the linkers 3 allows for and promotescross-linking with receptors. The multidimensional nature of thestructure illustrated in FIG. 1 was obtained using standard molecularmodeling techniques.

In a third aspect, the present invention provides a therapeuticcomposition, preferably pro-angiogenic. The composition preferablycomprises one or more of the therapeutic peptides disclosed herein, anda pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. There term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which an active ingredient isadministered. Such pharmaceutical carriers can be liquids, such as waterand oils, including those of petroleum, animal, vegetable or syntheticorigin. Preferably, the pharmaceutically acceptable carrier comprises,but is not limited to, a saline solution, a polyether, and/or water.Examples of suitable carriers, include water, phosphate-buffered saline,sodium chloride solutions, polyethylenelglycol solutions, etc. The typeand amount of carrier is typically influenced by the route ofadministration. For example, when the peptides are administered viainjection, preferable carriers comprise, but are not limited to, aphosphate-buffered saline solution having a pH between 6.5 and 7.5(e.g., about 7.2) or a sodium chloride solution (e.g., 100-150 mM);whereas when administered via a patch, the carrier preferably selectedfrom the group consisting of: polyethyleneglycol solution (e.g., 250mg/mL of PEG8000), carbopol gel base, propylene glycol, methyl paraben,ethyl paraben, HPMC gel base (hydroxypropylmethyl cellulose), PEG 4000,PEG 300, DMSO, isopropyl myristate, mineral oil, white petrolatum, beeswax, glycerine and water.

In a fourth aspect, the present invention provides a method formodulating the cytokine expression in a subject. Being able to modulatethe expression of endogenous cytokines provides a means of regulatingand treating a significant number of diseases. The method comprises thesteps of administering to the subject one or more of the therapeuticpeptides described herein, wherein the peptide is administered in anamount sufficient to increase or decrease the expression of at least oneendogenous cytokine in the subject. Preferably, the subject beingtreated by the method is an animal, more preferably a mammal, e.g.,monkey, dog, cat, horse, cow, sheep, pig, and most preferably thesubject is human.

In a preferred embodiment, the peptide modulates the expression of atleast one cytokine selected from the group consisting of: Eotaxin,Eotaxin-2, ICAM-1, 1-309, IL-4, IL-8, IL-10, IL-11, IL-15, IL-16, IL-17,IL-21, RANTES, sTNF RI, sTNF RII, IL-12p40, IL-12p70, M-CSF, MCP-2, MIG,PDGF-BB, TNF-β, MIP-1b, GCSF, and TIMP-2. More specifically, preferablythe peptides increase the endogenous expression of at least one cytokineselected from the group consisting of: IL-11, IL-12p40, IL-12p70,RANTES, sTNF RI, PDGF-BB, Eotaxin, Eotaxin-2, IL-15, IL-16, IL-17,MCP-2, M-CSF, MIG, TNF-β, sTNF RII, TIMP-2. Most preferably, the peptideincreases at least two, three, four, or more of these cytokines.Moreover, expression of these cytokines is preferably increased by atleast 20%, more preferably 50%, and most preferably 80%. Sometimes thebeneficial endogenous cytokine is increased by even more than 100%.

In addition, preferably the peptide decreases the endogenous expressionof at least one cytokine selected from the group consisting of: IL-7,IL-8, Ecotaxin-2, GCSF, ICAM-1, INF-γ, IL-6SR, TIMP-2, MCP-1, andMIP-1b. Likewise, expression of these cytokines is preferably decreasedby at least 20%, more preferably 50%, and most preferably 80%. Sometimesthe beneficial endogenous cytokine is decreased by even more than 100%.

In a further preferred embodiment, the peptide stimulates the release ofthose cytokines that induce angiogenesis. In another preferredembodiment, the peptide does not stimulate the release of thosecytokines that cause or exacerbate inflammation. In a specificembodiment, the peptide decreases at least one cytokine selected fromthe group consisting of: IL-1a, IL-8, IL-13, IL-11, IL-12p40, andIL-12p70. It is most preferable that the peptide does not stimulate therelease of IL-8. In this context, “does not stimulate” means levels ofIL-10 are not statistically greater (preferably p>0.20; more preferablyp>0.10; and most preferably p>0.05) between treatments and controlsamples when examined in experiments similar to those described inExample 3.

Preferably the “amount sufficient” as used herein, is the amountnecessary to modulate cytokine expression in a subject. In a morespecific embodiment, the amount sufficient is an amount within the rangeof 1 pmole to 1 nmole per g of body weight and/or within the range of0.1 to 300 mg per dose. For a typical adult human, the amount sufficientis usually within the range of 1 to 100 mg, more preferably, 1 to 70 mg,and most preferably 1 to 50 mg per dose. Based on the subject's bodyweight, preferably the amount is 0.01 to 1.4 mg/kg; more preferably 0.01to 1 mg/kg; and most preferably between 0.01 to 0.7 mg/kg of thesubject's body weight per dose. As a nonlimiting example, an amountsufficient to treat the disease in a typical 70 kg adult human would be0.1 mg/kg of the subject's body weight, 2 μmole, or 7 mg per dose. Aswould be known to one skilled in the art, the lifetime of activatedmacrophages suggests that a dose should be administered once about every2 to 6 days, more preferably 1 or 2 times a week, until the disease istreated, resulting in an improvement of at least one symptom, andpreferably by eradication from the body of the subject.

In a fifth aspect, the present invention provides a method forstimulating wound healing via induction of angiogenesis in a subject.The method comprises the steps of administering to the subject acomposition comprising a peptide of the invention, wherein the peptideis in an amount sufficient to stimulate angiogenesis in an areasubstantially near a subject's wound. An area substantially near asubject's wound” refers to the area of the body, preferably within 6inches, more preferably within 4 inches, and most preferably within 2inches from the outer edge of the wound being treated. Preferably, thesubject being treated by the method is an animal, more preferably amammal, and most preferably a human.

Administration of the composition to the subject comprises transferringthe composition into the body of the subject in an amount sufficient tostimulate wound healing via induction of angiogenesis. The compositionof the invention can be administered via any suitable route thatachieves the intended purpose. For example, administration can be bysubcutaneous, intravenous, intramuscular, intraperitoneal, buccal, orocular routes, rectally, parenterally, intrasystemically, topically (asby powders, ointments, drops or transdermal patch), or as an oral ornasal spray. Alternatively, or concurrently, administration can be bythe oral route. The dosage administered will be dependent upon the age,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

In a preferred embodiment, the composition is administered orally. Inthis embodiment, the composition is in an edible form, including forexample, powders, granules, capsules, pills, tablets, elixirs,suspensions, emulsions, syrups and the like. These preparations may besubjected to modification such as sustained-release, stabilization, easydisintegration, poor disintegration, enteric coating, easy absorptionand the like. Preferably in this embodiment, the composition is in aform that allows for passage through the stomach and release in theintestine for absorption in intestinal lumen, e.g., enteric coatedformations based on pH or timed release. Additionally, the dosage may bein the form of chewable preparations, sublingual preparations, buccalpreparations, troches, ointments, patches, solutions and the like. Thesepreparations may be also subjected to modification such assustained-release, stabilization, easy disintegration, poordisintegration, enteric coating, easy absorption and the like.

In another embodiment, the composition is administered via injection,e.g., subcutaneous, intramuscular, intravenous, and intraperitonealinjection, preferably subcutaneously. When the composition is formulatedfor transdermal administration, the composition preferably comprisesPEG8000, but may be comprised of other suitable carriers such ascarbopol gel base, propylene glycol, methyl paraben, ethyl paraben, HPMCgel base, PEG 4000, PEG 300, DMSO, isopropyl myristate, mineral oil,white petrolatum, bees wax, glycerine and water in a medical patch. Thepatch preferably comprises 1 to 8 mg, more preferably 2 to 6 mg, andmost preferably about 4 mg of therapeutic peptides per mL of solution inthe patch. A patch typically comprises 1 to 75 mL, and more preferably 1to 18 mL of solution within the patch. When administering to thesubject, the patch should be in contact with the subject's skin for aperiod of at least 2 to 72 hours. A typical patch would be in contactwith the subject's skin for approximately 24 to 48 hours.

As would be known by one skilled in the art, the PDGF class of cytokinesis instrumental in the process of angiogenesis. Thus, in a preferredembodiment, the stimulation of wound healing via the induction ofangiogenesis increases the production of cytokines comprising but notlimited to the PDGF class. In a further preferred embodiment, theadministration of the composition stimulates the production of thecytokine PDGF-BB. “Stimulates the production” in this instance meansPDGF-BB production is preferably increased by at least 20%, morepreferably 50%, and most preferably 80%. Data from TABLE 3 suggestsPDGF-BB production may increase by at least 90%.

Preferably the administration of the compositions of the invention doesnot cause inflammation in the subject. As would be known by one skilledin the art, inflammation in response to a wound is often caused by therelease of the cytokines IL-8 and ICAM-1 in the body. Thus, in a furtherpreferred embodiment, the administration of the composition inhibits theproduction of cytokines that cause inflammation or at least does notincrease the production of these cytokines. More preferably, theadministration of the composition inhibits the production of IL-8. Incertain embodiment, the composition actually promotes the release and/orproduction of anti-inflammatory cytokines.

As would be known by one skilled in the art, selective cross-linking ofcell-surface receptors by a multivalent structure incorporating at leastone peptide embodiment of the present invention may attenuate bacterialinfections by stimulating activity of phagocytic cells that arerecruited to the injured or infected tissue. Phagocytic cells respond tothe presence of bacterial cells containing lipopolysaccharide (LPS) ontheir surface by engulfing the cells into phagocytic vacuoles anddigesting the bacterial cells. In addition, the phagocytic cells respondto the presence of antibodies directed toward—and bound to—a pathogensuch as a bacterial cell, fungal cell or virus. Thus, in anotherpreferred embodiment, the administration of the composition enhances theability of the subject's immune system to ward off or attenuateinfection. In a most preferred embodiment, the infection is attenuatedor prevented at or substantially near the target wound area. An areasubstantially near a subject's wound” refers to the area of the body,preferably within 6 inches, more preferably within 4 inches, and mostpreferably within 2 inches from the outer edge of the wound beingtreated.

EXAMPLES

Elements and acts in the examples are intended to illustrate theinvention for the sake of simplicity and have not necessarily beenrendered according to any particular sequence or embodiment. Theexamples are further intended to establish possession of the inventionby the Inventors.

Example 1 Peptide Design and Synthesis

A screen of peptide sequences identified one set of sequences ofinterest. The corresponding peptides were synthesized by solid-phasemethods using standard Fmoc side chain protection. Branched peptideswere constructed on a central tri-lysine framework, which allows fouridentical sequences within the same structure. A (Gly)₃-Ser (GGGS, SEQID NO:3) linker sequence was included to distance the active sequencefrom the central framework. Distances between the active sequences canbe adjusted by decreasing or increasing the length of the linker,including without limitation the use of two linkers in tandem (GGGSGGGS,SEQ ID NO:4) or by inserting any inert linker such as polyethyleneglycol (PEG) of a variable length. The branched structure was designedto have enhanced activity by causing receptor clustering (cross-linking)on the surface of responsive cells.

The peptides were synthesized on PAL-PEG-polystyrene resin (AppliedBiosystems, Foster City, Calif.) utilizing Fmoc(9-fluorenylmethoxycarbonyl)-protected amino acids and a MilligenBiosearch 9050+ continuous flow peptide synthesizer (MilliporeCorporation, Billerica, Mass.).

The C-terminus of the central framework is typically a lysine residuecontaining an amide derivative of the carboxyl group. However, theC-terminus can be modified to include additional C-terminal amino acidssuch as a cysteine residue, to which tags such as fluorescent groups canbe added, or an ϵ-biotinyl-N-lysine (biotinyl-K) residue useful forsubsequent purification processes. In addition, an amino acid such asβ-alanine (βA) or tryptophan can be inserted between the addedC-terminal amino acid and the C-terminal lysine residue of the centralframework in order to provide a spacer or a means to determineconcentration by absorbance. Non-limiting examples of such modifiedC-terminal lysine residues on the central framework include K-βA-C andK-W-biotinyl-K, respectively. Furthermore, additional lysine residuescan be added to either one or both of the α- and ϵ-amino groups of amodified C-terminal lysine on the central framework to yield, forexample, (K)₂K, (K)₂K-βA-C or (K)₂K-W-biotinyl-K, thereby formingbranched structures in which the α- and ϵ-amino groups are available forextension.

The lysine residues used at the branch points are incorporated with Fmocprotection on both the α- and ϵ-amino groups, so that both becomeavailable for amide bond formation after the standard deprotectionreaction with piperidine. A dansyl group (or other fluorescent tag) maybe incorporated by reaction with the thiol group on the C-terminalcysteine residue using 5-((((2iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS)following a standard protocol for thiol-reactive probes (InvitrogenCorp., Carlsbad, Calif.). Biotin is attached to lysine through an amidelinkage to the side chain amino group which, because of its highaffinity with streptavidin, provides a means to retrieve the peptidewith associated proteins from reaction mixtures in order to study theinteraction of the peptide with cellular components.

The peptides were built attached to a solid-phase resin, which waschosen such that when the peptide is cleaved from the resin, thecarboxyl group at the C-terminus of the peptide is released as theamide. Each of the four amino groups of the tri-lysine central frameworkwas extended by addition of the linker, GGGS (SEQ ID NO:3), followed bythe core sequence.

After cleavage from the resin bed, the product can be substantially orcompletely purified by HPLC on a preparative Jupiter Proteo C12 column(21.2 mm×250 mm) (Phenomenex, Inc., Torrance, Calif.) using a gradientfrom 8% to 18% acetonitrile in water containing 10 mM trifluoroaceticacid (TFA). The purity of the final peptide product was confirmed bymass spectroscopy performed using a Voyager DE STR mass spectrometer(Applied Biosystems, Foster City, Calif.). HPLC-purified peptide wasdried under vacuum, dissolved in sterile phosphate buffered saline, pH7.2 (PBS) and passed through a gel filtration column of Sephadex G 15 orG 25 (1×48 cm for small samples) to separate TFA from the peptide. Thecolumn is then eluted with sterile PBS. Endotoxin is removed by passageof the peptide through a DEAE-Sephadex A-25 column.

Alternatively, the product is purified by use of a C18 reverse-phasecartridge, ion exchange chromatography, and gel filtrationchromatography to remove side products of synthesis. Concentration canbe determined by absorbance of the fluorophore (e.g., dansyl group,extinction coefficient, ϵ_(mM)=5.7 cm⁻¹ at 336 nm), absorbance of thepeptide bond at 210 nm (ϵ_(mg/mL)≈31 cm⁻¹), absorbance of aromatic aminoacids (e.g., tryptophan, ϵ_(mM)=5.6 cm⁻¹ at 280 nm) in the peptide (whenpresent) and/or absorbance of the bicinchoninic acid reagent (PierceBiotechnology, Rockford, Ill.). The peptide solutions can be adjusted tothe desired concentration and filter-sterilized prior to use.

Example 2 Core Peptide Sequences: WNSTL (SEQ ID NO:1) and NQHTPR (SEQ IDNO:2)

The peptides (WNSTL, SEQ ID NO:1) and (NQHTPR, SEQ ID NO:2) wereidentified through a screen of peptide sequences as potentially ofinterest. The sequences were synthesized on a tri-lysine core accordingto Posnett et al., J. Biol. Chem., 263: 1719-25, 1988, with a linker(GGGS, SEQ ID NO:3) included with the sequence to extend the activepeptide away from the core.

FIGS. 2A-B illustrate the chemical structure of these two embodiments ofthe invention. In these embodiments, R═H or can be an adduct containinga fluorescent tag such as the dansyl group shown in FIG. 3. The peptideconstructs illustrated contain four identical sequences, each of whichis connected to a branched central tri-lysine framework via a (Gly)₃Ser(GGGS, SEQ ID NO:3) linker. FIG. 2A is a branched peptide constructaccording to one embodiment of the invention, a peptide construct whichcontains four copies of the core sequence WNSTL (SEQ ID NO:1). Thepeptide embodiment containing R═H has a molecular mass of 3,841.16Daltons.

FIG. 2B illustrates the structure of the construct containing fourcopies of the N-terminal core sequence NQHTPR (SEQ ID NO:2). Thispeptide embodiment containing R═H has a molecular mass of 4,543.89. Inanother embodiment, a C-terminal extension containing β-alanine,cysteine and a dansyl tag, as shown in FIG. 3, is covalently added tothe construct illustrated in FIG. 2B, resulting in a peptide having amolecular mass of 4,850.23 Daltons.

Example 3 Regulation of Cytokine Release

To determine whether the peptides regulate induction or inhibition ofrelease of cytokines, cultured peripheral blood mononuclear cells(PBMCs) were treated with one peptide embodiment of the invention and,after 4 hour incubation, the medium was collected and assayed forchanges in the amounts of 40 different cytokines. The peptide constructcontaining four copies of WNSTL (SEQ ID NO:1), illustrated in FIG. 2A,was added at a concentration of 100 nM in each of the assays. The PBMCcultures were established with cells from Cellular Technology, Ltd.(Shaker Heights, Ohio). Approximately 3 million cells of frozen humanPBMCs were thawed at 37° C. and transferred to a 50 mL conical tubewhere 8 mL of wash medium were added slowly. Then an additional 8 mLwere added and swirled to mix. The cells were then centrifuged at 330 gfor 10 min, the supernatant was removed and the pellet was resuspendedin 10 mL wash medium and centrifuged as above. The washed cells werethen resuspended in RPMI 1640 medium containing 10% FBS to about 6million cells per mL and 100 μL of the suspension were added into eachwell of a 96-well microtiter plate and incubated overnight at 37° C. inhumidified 5% CO₂. After 24 hr, the medium was replaced with 200 μLfresh RPMI 1640 medium containing 10% FBS and incubated at 37° C. inhumidified 5% CO₂ for 2 days. For the data shown in TABLE 1, an aliquotof the peptide construct was added to samples in duplicate at a finalconcentration of 5 nM or 100 nM and incubated at 37° C. in humidified 5%CO₂ for 4 hr. For other experiments (data not included), the incubationwas continued for 24 hr. The medium was then removed and stored at 80°C. The samples were analyzed for production of cytokines. One set ofcontrol cells was not treated with an experimental agent. A second setof control cells was treated with LPS, the agent commonly used to induceproduction of a variety of inflammatory cytokines. The positive controlfor inflammation provided by this second set of control cells wasessential to ensure that the peptides do not produce an unregulatedinflammatory response.

Assays of cytokine levels in samples of culture media were performedusing methods developed by RayBiotech, Inc. (Norcross, Ga.). In thistechnology, membrane arrays of antibodies against cytokines wereincubated with samples of media. After washing, the array was incubatedwith a cocktail of all antibodies tagged with biotin. The membrane wasthen washed free of unbound antibodies and incubated with streptavidinlabeled with a fluorescent dye. After a final wash, the membrane arrayswere read in a fluorescence detector and the intensities of the spotsquantitated to obtain relative values.

TABLES 1-3 list a number of cytokines whose concentrations in a mediumof PBMC cultures can be altered as the result of treatment of the cellswith peptide embodiments of the present invention. The cytokines thusaffected include without limitation:

Eotaxin (chemoattractant, induces substantial accumulation ofeosinophils);

Eotaxin-2 (induces chemotaxis of eosinophils and basophils, release ofhistamine);

GCSF (granulocyte colony stimulating factor, growth factor);

ICAM-1 (intercellular adhesion molecule-1, binds to integrins, humanrhinovirus receptor);

IL-1β (Interleukin 1β, a mediator of inflammatory reactions);

IL-4 (promotes proliferation and differentiation of B-cells and inhibitsproduction of inflammatory cytokines such as IL-1, IL-6 and TNF-α);

IL-6SR (soluble receptor for IL-6);

IL-7 (stimulates proliferation of precursor B and T cells);

IL-8 (chemoattractant and activator of neutrophils);

IL-10 (inhibits synthesis of inflammatory cytokines such as INF-γ, IL-2and TNF-β);

IL-11 (induces inflammatory responses, promotes immune responses);

IL-12 (contains subunits of 40 and 70 kDa, activates NK-cells andstimulates proliferation of lymphoblasts);

IL-15 (has many of the same properties as IL-2, may contribute to T-cellmediated immune responses);

IL-16 (chemoattractant and activator for cells that express CD4);

IL-17 (functions as a mediator of angiogenesis that stimulates migrationof vascular endothelial cells and cord formation and regulatesproduction of a variety of growth factors promoting angiogenesis);

INF-γ (Interferon-gamma, has antiviral, immunoregulatory and anti-tumorproperties);

MCP-1,2 (monocyte chemotactic proteins);

M-CSF (induces proliferation and stimulates monocytes and macrophages);MIG (chemoattractant for stimulated T cells but not active onneutrophils or monocytes);

MIP-1b (macrophage inflammatory protein, involved in cell activation ofgranulocytes and killer cells);

PDGF-BB (platelet-derived growth factor, BB homodimer);

RANTES (regulated on activation, normal T cell expressed, and secreted;chemotactic for T cells);

sTNF RI, Rh (soluble forms of receptor RI or RII for tumor necrosisfactor (TNF));

TIMP-2 (tissue inhibitor of metalloproteinases of the extracellularmatrix); and

TNF-β (promotes the proliferation of fibroblasts and is involved inwound healing).

TABLE 1 contains data showing cytokines that are released at asignificantly higher or lower rate (compared to untreated controls)during a 4-hour incubation of PBMCs with the branched peptide constructcontaining four copies of WNSTL (SEQ ID NO:1) in the presence of serum.One set of control samples was not treated with peptide and a second setof control samples was treated with lipopolysaccharide (LPS) in theabsence of the peptide construct. The structure of the construct isillustrated in FIG. 2A. Among the cytokines that are induced to morethan two-fold higher concentrations as a result of incubation with theconstruct containing four copies of WNSTL (SEQ ID NO:1) are PDGF-BB,IL-1β, IL-4, IL-1, IL-12 and RANTES. In contrast, several cytokines showmore than a two-fold decrease in concentration as compared to untreatedcontrol samples with this peptide. The decrease in IL-8 concentration inthe peptide-treated sample, as compared with the amount of IL-8 in thesample treated with the proto-typical inflammatory agent LPS, isparticularly notable. Furthermore, when compared to untreated controlsamples, the peptides did not induce a change in the amount of theinflammatory cytokine IL-6. However, IL-6 levels were significantlyelevated in samples treated with LPS in the absence of peptide. In oneexperiment, for example, the peptide treated sample had a relative IL-6concentration of 93, the untreated control sample, 98, and theLPS-treated sample (in the absence of peptide), 5,879. Because theconcentration of IL-6 in the peptide treated sample was notsignificantly different from the untreated control sample, the data forIL-6 are not included in TABLES 1 or 2. The increase in theproangiogenic PDGF-BB and the decrease in the inflammatory IL-8, withoutconcomitant stimulation of inflammation, are of particular importancewith respect to wound healing.

TABLE 1 Relative Cytokine Concentration after Incubation of PBMCs inSerum with a Peptide Construct Containing Four Copies of the CoreSequence WNSTL (SEQ ID NO: 1). WNSTL Cytokine (SEQ ID NO: 1) Untreated(control) LPS (control) Increased: PDGF-BB 159 43 56 IL-1β 120 44 47IL-4 97 30 49 IL-11 49 17 26 IL-12p40 228 108 46 IL-12p70 131 90 89RANTES 145 80 114 STNF RI 78 42 58 Decreased: IL-7 93 154 178 IL-8 144417 840 IL-10 43 101 218 Eotaxin-2 109 1934 469 GCSF 50 108 120 ICAM-144 57 53 INF-γ 103 134 158 IL-6sR 32 52 52 TIMP-2 25 58 92 MCP-1 9681464 1844

Relative cytokine concentration data for the peptide constructcontaining four copies of the core sequence NQHTPR (SEQ ID NO:2) issimilarly outlined in TABLE 2. The structure of the construct isillustrated in FIG. 2B. Cytokines were again observed at significantlyhigher or lower concentrations (relative to controls) after a 4-hourincubation with the branched construct containing four copies of thecore sequence NQHTPR (SEQ ID NO:2). The data in TABLE 2 show that,although the overall pattern of cytokine response to this peptide issomewhat different from that of the quadravalent peptide constructcontaining the core sequence WNSTL (SEQ ID NO:1) shown in TABLE 1, itsimilarly induces a higher amount of PDGF and a lower amount of IL-8.

TABLE 2 Relative Cytokine Concentration after Incubation of PBMCs inSerum with the Peptide Construct Containing Four Copies of the CoreSequence NQHTPR (SEQ ID NO: 2). Cytokine NQHTPR (SEQ ID NO: 2) Untreated(control) Increased: PDGF-BB 84 43 Eotaxin 57 32 Eotaxin-2 310 193 IL-15146 97 IL-16 8 1 IL-17 14 5 MCP-2 90 56 M-CSF 116 44 MIG 100 54 TNF-β 8438 sTNF RII 26 8 TIMP-2 110 58 Decreased: IL-8 261 417 MIP-1b 777 1172

TABLE 3 is based on the same data as TABLES 1-2, and shows the effectsof constructs containing four copies of VQATQS (SEQ ID NO:8), VSNQH (SEQID NO:9), NQHTPR (SEQ ID:2) and WNSTL (SEQ ID NO:1) on the relativeconcentrations of cytokines in PBMCs treated for four hours with eachpeptide construct as compared with untreated control cultures andLPS-treated cells.

TABLE 3 Relative Concentrations of Cytokines in PBMCs Treated for 4 hrwith each Peptide Construct as Compared with Untreated Control Culturesand LPS-treated Cells.* Core Sequence of Peptide Cytokine VQATQS VSNQHNQHTPR WNSTL Untreated LPS Eotaxin 47 57 32 31 Eotaxin-2 401 470 310 109193 469 GCSF 50 108 120 GM-CSF 96 57 106 ICAM-1 70 44 57 53 IFN-γ 170103 134 158 I-309 101 26 39 IL-1α 322 225 246 IL-1β 120 44 47 IL-2 86 90IL-3 130 132 IL-4 64 97 30 49 IL-6 167 98 4,375 IL-6sR 32 52 52 IL-7 93154 178 IL-8 261 144 417 840 IL-10 67 43 101 218 IL-11 49 17 26 IL-12p4074 52 45 228 108 46 IL-12p70 112 117 65 131 90 89 IL-13 138 125 IL-15134 137 146 97 104 IL-16 10 8 1 2 IL-17 21 21 14 5 10 IL-21 90 130 200 —50 (IFN-γ: 100) IP-10 334 377 230 268 MCP-1 1,966 968 1,464 1,844 MCP-297 90 56 177 M-CSF 90 116 44 59 MIG 94 100 54 66 MIP-1a 81 237 MIP-1b777 1,172 1,828 MIP-1d 40 38 RANTES 145 80 114 TGF-β1 83 62 62 TNF-α 98138 73 93 TNF-β 91 93 84 38 95 sTNF RI 75 78 42 58 sTNF RII 20 26 8 26PDGF-BB 83 82 84 159 43 56 TIMP-2 175 285 110 25 58 92 *The absence of anumber indicates no significant change from untreated control cultures.

Toxicity of the peptide in vivo can be tested by injection of a peptideinto animal subjects, including without limitation mice. The peptidescan be administered in a number of ways, including without limitation byinjection (intravenously, subcutaneously, intramuscularly orintraperitoneally), topically (transmucosally, transbuccally, ortransdermally) and/or orally (liquid, tablet or capsule). In preliminarystudies on mice, no adverse effects of the peptide on the growth rate ofthe animals have been observed after injection of an effective dose onalternate days for 1 month (data not shown).

Example 4 Pro-Angiogenic Activity of Peptides

Tumors require vascularization to obtain nutrients to support growth.Therefore, stimulation of growth of a tumor in response toadministration of a construct of this invention is an indication ofangiogenesis. For this example, a xenograph model system with the nudemouse (nu/nu) was used to determine the effect of peptide on growth of786-0 human renal cell adenocarcinoma cell line injected into the flankof a mouse so as to induce a tumor. After the tumor was established,peptide was injected subcutaneously on alternate days. The weight of thetumor was estimated by calculation of the volume.

FIG. 4 illustrates data resulting from assays for pro-angiogenicactivity for one embodiment of the invention. The bar graph in FIG. 4shows the average weight of the tumor in mice treated with the peptideconstruct containing four copies of the core sequence WNSTL (SEQ IDNO:1) at a dose of 0.05 nmole/gm as compared with a control group. Thepeptide was also assayed in combination with Sorafenib, ananti-angiogenic drug. The results shown in FIG. 4 indicate that growthof the tumor was significantly enhanced, by a factor of 1.7, over thecontrol in mice to which the construct with four copies of the coresequence WNSTL (SEQ ID NO:1) was administered. The drug Sorafenib, aninhibitor of angiogenesis, strongly (but not completely) inhibited theeffect of the peptide.

Example 5 Stimulation of Phagocytosis by Peptides

The activity of the peptides to stimulate phagocytosis was assessed bythe ability of macrophages to engulf microspheres opsonized withanti-HIV antibodies. A biotin-tagged peptide epitope of a surfaceprotein of HIV-1 was bound to streptavidin on the surface of the beads.An antibody preparation that was raised against this epitope was thenbound to the HIV peptide. The beads were then washed and presented toPBMC cultures pretreated with peptides. Macrophages in cultures nottreated with peptides had little, if any, phagocytic activity. Inmultiple control cultures, the number of beads within a macrophage-likecell ranged from 0 to 3. In the cultures treated with peptide constructscontaining four copies of the peptide sequences WNSTL (SEQ ID NO:1),NQHTPR (SEQ ID NO:2), or VQATQS (SEQ ID NO:8), an average of 10 beadswere counted in each phagocytic cell, with no substantial differencebetween peptide constructs. These findings suggest a stimulation ofphagocytic activity by the peptide constructs compared to untreatedcultures. Cultures treated with the cytokine INF-γ exhibited an averageof roughly 15 beads per phagocytic cell.

Example 6 Devices or Materials Containing the Peptides

The peptides of the present invention may also be deposited to providean appropriate coating to a surface, including without limitationbioactive surfaces or inert, non-biological surfaces of a device ormaterials designed for implantation. The peptides can thus promotehealing around the implanted materials in order to achievevascularization without scarring. They could similarly be used in otherin vitro or in vivo applications, including without limitation, withembedded sensors.

The embodiments and examples set forth herein were presented in order tobest explain the present invention and its practical application and tothereby enable those of ordinary skill in the art to make and use theinvention. Although the examples herein disclose the therapeuticefficacy of the peptides of the present invention with respect to woundhealing, for example, the peptides may also be useful for restoration ofcirculation generally, including circulation compromised by chronicconditions such as diabetes, circulation to damaged tissues, and othersimilar disorders. Furthermore, the use of larger peptides containingactive core sequences could potentially enhance the therapeutic benefitsdisclosed herein.

What is claimed is:
 1. A therapeutic peptide consisting of between 5 to6 amino acids and a peptide sequence selected from the group consistingof: X1-Q-X2-X3-X4-X5; and X1-N-S-X3-X4-X5 wherein X1 is selected fromthe group consisting of N, V, W, and Y; X2 is selected from the groupconsisting of H, A, and P; X3 is selected from the group consisting ofT, Q, and S; X4 is selected from the group consisting of P, Q, H, L, andY; and X5 is selected from the group consisting of R and S, or isabsent.
 2. The therapeutic peptide of claim 1, in a substantially pureform of at least 80% by weight.
 3. The therapeutic peptide of claim 1,wherein the N-terminus is acetylated and the peptide is pro-angiogenic.4. A therapeutic composition comprising the therapeutic peptide of claim1 and a pharmaceutically acceptable carrier.
 5. A method of modulating acytokine expression in a subject, the method comprising administering toa subject one or more therapeutic peptides of claim 1, wherein thetherapeutic peptide is administered in an amount sufficient to modulatethe expression of at least one endogenous cytokine.
 6. The method ofclaim 5, wherein the at least one cytokine is selected from the groupconsisting of: Eotaxin, Eotaxin-2, ICAM-1, 1-309, IL-4, IL-8, IL-10,IL-11, IL-15, IL-16, IL-17, IL-21, RANTES, sTNF RI, sTNF RII, IL-12p40,IL-12p70, M-CSF, MCP-2, MIG, PDGF-BB, TNF-β, MIP-1b, GCSF, and TIMP-2.7. The method of claim 5, wherein the therapeutic peptide increases theendogenous expression of at least one cytokine selected from the groupconsisting of: IL-11, IL-12p40, IL-12p70, RANTES, sTNF RI, PDGF-BB,Eotaxin, Eotaxin-2, IL-15, IL-16, IL-17, MCP-2, M-CSF, MIG, TNF-β, sTNFRII, and TIMP-2; and/or decreases the endogenous expression of at leastone cytokine selected from the group consisting of: IL-7, IL-8, IL-10,Eotaxin-2, GCSF, ICAM-1, INF-γ, IL-6sR, TIMP-2, MCP-1, and MIP-1b.
 8. Amethod of stimulating wound healing in a subject via induction ofangiogenesis, the method comprising: administering the composition ofclaim 4 to the subject in an amount sufficient to stimulate angiogenesisof a wound area.
 9. The method of claim 8, wherein the method comprisesmodulating the release of a profile of cytokines in the subject,comprising increasing the release in the subject of at least onetherapeutically beneficial cytokine and/or inhibiting in the subject theproduction or release of at least one therapeutically deleteriouscytokine.
 10. The method of claim 9, further comprising stimulating theactivity of phagocytic cells and modulating the immune system of thatsubject to further promote wound healing and decrease inflammation of awound area.
 11. The method of claim 9, wherein the cytokine profilereleased is indicative of a reduction of inflammation.
 12. The method ofclaim 9, wherein the at least one beneficial cytokine is PDGF-BB and theat least one deleterious cytokine is IL-8.
 13. The method of claim 9,wherein the administration of the composition enhances the ability ofthe immune system to ward off or attenuate infection at the wound. 14.The method of claim 9, wherein the amount is within the range of 1 pmoleto 1 nmole per g of body weight of the subject.
 15. The method of claim9, wherein the amount administered is within the range of 0.1 to 300 mgper dose, and the dose is administered one or two times per week. 16.The method of claim 9, wherein the composition is in a formulationsuitable for oral administration to the subject, the dosage designed topass through the subject's stomach before releasing the peptide in thesubject's intestine for absorption through the intestinal epithelium.17. The method of claim 9, wherein the composition is administered via apatch comprising: polyethyleneglycol, propylene glycol, methyl paraben,ethyl paraben, hydroxypropylmethyl cellulose, DMSO, isopropyl myristate,mineral oil, white petrolatum, bees wax, or glycerine.
 18. A therapeuticcomposition comprising: a pharmaceutically acceptable carrier; and atherapeutic peptide having the peptide sequence of VSNQH (SEQ ID NO:9)or a polypeptide comprising a construct having a central framework andat least two arms consisting of a core sequence having the peptidesequence of VSNQH (SEQ ID NO:9), wherein the core sequence is linked tothe central framework via a linker.
 19. The therapeutic composition ofclaim 18, wherein the central framework consists of a tri-lysine coreand the linker is selected from the group consisting of GGGS (SEQ IDNO:3), GGGSGGGS (SEQ ID NO:4), SSSS (SEQ ID NO:10), and SSSSSSSS (SEQ IDNO:11).
 20. The therapeutic composition of claim 18, wherein thepolypeptide comprises four arms.
 21. The therapeutic composition ofclaim 18, wherein the therapeutic peptide has at least two armsconsisting of a core sequence having the peptide sequence of VSNQH (SEQID NO:9) and wherein the core sequence is linked to the centralframework via a linker.
 22. The therapeutic polypeptide of claim 21,wherein the central framework consists of a tri-lysine core and thelinker is selected from the group consisting of GGGS (SEQ ID NO:3),GGGSGGGS (SEQ ID NO:4), SSSS (SEQ ID NO:10), and SSSSSSSS (SEQ IDNO:11).
 23. The therapeutic composition of claim 18, wherein thepolypeptide comprises eight arms.
 24. A therapeutic peptide having apeptide sequence of VSNQH (SEQ ID NO:9) or a polypeptide comprising aconstruct having a central framework and at least two arms consisting ofa core sequence having the peptide sequence of VSNQH (SEQ ID NO:9),wherein the core sequence is linked to the central framework via alinker.
 25. The therapeutic peptide of claim 24, in a substantially pureform of at least 80% by weight.
 26. The therapeutic peptide of claim 24,wherein the N-terminus is acetylated and the peptide is pro-angiogenic.27. A method of modulating a cytokine expression in a subject, themethod comprising administering to a subject one or more therapeuticpeptides of claim 24, wherein the therapeutic peptide is administered inan amount sufficient to modulate the expression of at least oneendogenous cytokine.
 28. The method of claim 27, wherein the at leastone cytokine is selected from the group consisting of: Eotaxin,Eotaxin-2, ICAM-1, 1-309, IL-4, IL-8, IL-10, IL-11, IL-15, IL-16, IL-17,IL-21, RANTES, sTNF RI, sTNF RII, IL-12p40, IL-12p70, M-CSF, MCP-2, MIG,PDGF-BB, TNF-β, MIP-1b, GCSF, and TIMP-2.
 29. The method of claim 27,wherein the therapeutic peptide increases the endogenous expression ofat least one cytokine selected from the group consisting of: IL-11,IL-12p40, IL-12p70, RANTES, sTNF RI, PDGF-BB, Eotaxin, Eotaxin-2, IL-15,IL-16, IL-17, MCP-2, M-CSF, MIG, TNF-β, sTNF RII, and TIMP-2; and/ordecreases the endogenous expression of at least one cytokine selectedfrom the group consisting of: IL-7, IL-8, IL-10, Eotaxin-2, GCSF,ICAM-1, INF-γ, IL-6sR, TIMP-2, MCP-1, and MIP-1b.
 30. A method ofstimulating wound healing in a subject via induction of angiogenesis,the method comprising: administering the composition of claim 18 to thesubject in an amount sufficient to stimulate angiogenesis of a woundarea.
 31. The method of claim 30, wherein the method comprisesmodulating the release of a profile of cytokines in the subject,comprising increasing the release in the subject of at least onetherapeutically beneficial cytokine and/or inhibiting in the subject theproduction or release of at least one therapeutically deleteriouscytokine.
 32. The method of claim 31, further comprising stimulating theactivity of phagocytic cells and modulating the immune system of thatsubject to further promote wound healing and decrease inflammation of awound area and/or the cytokine profile released is indicative of areduction of inflammation and the at least one beneficial cytokine isPDGF-BB and the at least one deleterious cytokine is IL-8.
 33. Themethod of claim 31, wherein the administration of the compositionenhances the ability of the immune system to ward off or attenuateinfection at the wound.
 34. The method of claim 31, wherein the amountis within the range of 1 pmole to 1 nmole per g of body weight of thesubject.
 35. The method of claim 31, wherein the amount administered iswithin the range of 0.1 to 300 mg per dose, and the dose is administeredone or two times per week.
 36. The method of claim 31, wherein thecomposition is in a formulation suitable for oral administration to thesubject, the dosage designed to pass through the subject's stomachbefore releasing the peptide in the subject's intestine for absorptionthrough the intestinal epithelium.
 37. The method of claim 31, whereinthe composition is administered via a patch comprising:polyethyleneglycol, propylene glycol, methyl paraben, ethyl paraben,hydroxypropylmethyl cellulose, DMSO, isopropyl myristate, mineral oil,white petrolatum, bees wax, or glycerine.